Fibrin deposits form at sites of vascular injury including ruptured atherosclerotic plaques. The initial event leading to fibrin formation is the activation of the extrinsic coagulation pathway which is triggered by the contact of tissue factor with circulating factor VII/VIIa. Tissue factor is a membrane-bound regulatory protein present outside blood vessels as well as in atherosclerotic plaques. When tissue factor is exposed to blood by vascular injury or plaque rupture, it binds to factor VII/VIIa in the presence of Ca.sup.++. The complex of tissue factor and factor VII/VIIa activates factor X to factor Xa, which in turn, activates prothrombin to thrombin in the presence of factor Va, phospholipid and Ca.sup.++. The resulting thrombin converts soluble fibrinogen to insoluble fibrin which deposits as clots.
The complex of tissue factor and factor VII/VIIa not only activates factor X but also activates factor IX to form factor IXa. Factor IXa activates factor X in the presence of phospholipid, factor VIIIa and Ca.sup.++. The reactions, except for the conversion of fibrinogen to fibrin, require negatively charged phospholipid, such as phosphatidylserine (PS), for optimal catalysis and proceed at the surface of insoluble phospholipid to localize fibrin clot formation. Thus, major components of fibrin clots are insoluble fibrin, phospholipid and the activated coagulation factors.
Phosphatidylserine, which is highly thrombogenic in vitro, generally is absent from the external face of the plasma membrane in both erythrocytes and platelets. The asymmetry is maintained by an active transport mechanism. It is believed that the asymmetrical distribution of PS is altered by platelet activation and PS becomes exposed on the external face of the plasma membrane. That reorientation provides negatively charged phospholipids for the formation of the prothrombinase complex and also enhances other phospholipid-dependent reactions in the coagulation cascade.
The coagulation factors that contain gamma-carboxyglutamic acid residues (factors X, IX, VII and prothrombin) bind to negatively charged phospholipids with banding constants in the 10.sup.-6 -10.sup.-7 M range in the presence of Ca.sup.++. The major source of negatively charged phospholipids for blood coagulation in vivo is thought to be the platelet plasma membrane. In fact, factor Xa binds to the surface of activated platelets where it forms a complex (prothrombinase) with platelet-bound factor Va to activate prothrombin.
Plasmin, a serine protease, is the sole plasma enzyme responsible for fibrin dissolution. It circulates in blood as a precursor, plasminogen. Plasminogen is a single-chain polypeptide that is converted to the two-chain active form, plasmin, by plasminogen activators. Plasmin is composed of an NH.sub.2 -terminal A-chain and a COOH-terminal B-chain held together by a disulfide bond. The A-chain contains five characteristic repeating units (kringle domains) while the B-chain contains the serine-protease catalytic unit. The region from the first kringle through fourth kringle binds to fibrinogen and some of the circulating plasminogen coprecipitates with fibrin when a clot is formed. Plasminogen that is proteolytically cleaved (Lys-plasminogen) has a higher affinity for fibrin clots than intact plasminogen (Glu-plasminogen) and thus accelerates lysis of fibrin clots. The fibrin binding sites of plasminogen are located in the first and fourth kringle domains.
Tissue-type plasminogen activator (tPA) and urokinase (uPA) are two physiologic activators of plasminogen. Both activators are synthesized as single-chain zymogens and are converted into two-chain active forms. tPA is a membrane bound protein synthesized mainly in endothelial cells and released into the blood stream in response to certain stimuli. The secretion of tPA into the blood stream triggers extrinsic fibrinolysis. The NH.sub.2 -terminal chain of tPA contains a finger domain, a growth factor-like domain and two kringle domains. The second kringle domain has binding affinity (K.sub.d, also known as binding constant or affinity constant) for the fibrin clot of 1.6.times.10.sup.-7 M. The catalytic efficiency of tPA is about 1,000 times higher toward fibrin-bound plasminogen than circulating plasminogen.
Prourokinase (single-chain urokinase or scuPA), a precursor of urokinase, is present in blood at low concentrations. Prourokinase is activated by plasma kallikrein and plasmin to stimulate intrinsic fibrinolysis. Although scuPA has a kringle domain, it shows little binding affinity for fibrin clots. However, it hydrolyzes fibrin-bound plasminogen more efficiently than free plasminogen.
A bacterial protein, streptokinase (SK), forms a stoichiometric complex with plasminogen which converts plasminogen to plasmin.
Fibrinolytic agents, tPA, uPA, scuPA and SK are being used as therapeutic agents to treat patients suffering from thrombosis. Although the agents represent a major advance, problems remain due to short half-life in circulation and a propensity to cause systemic fibrinogenolysis. For example, the relatively insufficient binding affinity for fibrin and probable cross-binding to circulating fibrinogen force a high-dose administration of tPA, which causes a significant degree of fibrinogenolysis.
The above therapeutic proteins have been modified in attempts to overcome the above-noted problems. The strategies used for improvements include: making the molecules resistant to circulating inhibitors; strengthening binding affinity for fibrin clots; and targeting fibrin deposits by conjugating plasminogen activator with antibody specific to fibrin clots.
Acylated plasminogen/SK (APS) has a higher fibrin selectivity than SK. The APS conjugate is not inactivated by a .alpha..sub.2 -plasmin inhibitor because the hydroxygroup of the active site serine residue is blocked. APS binds to fibrin clots and its acyl group is cleaved slowly to produce the active form. The half-life of APS is significantly longer than that of the unmodified parent molecule.
A truncated scuPA (residues 1-143 deleted) which lacks the growth factor and kringle domains in the heavy chain was expressed. The molecule has a fibrin selectivity identical to the intact form but is not inhibited by plasminogen activator inhibitor-1 (PAI-1),
A modified tPA where the binding site (residues 296-302) to PAI-1 was deleted by site-directed mutagenesis was also expressed, The molecule has the same enzyme activity as the native tPA but had a strong resistance to inhibition by PAI-1 and other setpins in circulating blood,
A chimeric molecule that combined the fibrin binding domain of plasminogen (A-chain) with the catalytic domain of urokinase had a eight-fold higher binding affinity for fibrin clots than urokinase. It also had a higher catalytic activity toward the fibrin monomer.
A chimeric molecule composed of the A chain of plasminogen and the catalytic domain of tPA had the same binding affinity for fibrin as plasminogen and the same catalytic activity as native tPA.
Murine monoclonal antibodies specific to the beta-chain of fibrin were conjugated with the catalytic chain of tPA or scuPA using a disulfide cross-linking agent. The antibodies have dissociation constants on the order of 2.times.10.sup.-7 M. The antibody/tPA conjugate was ten times more active than native tPA in lysis of the fibrin monomer. An antibody/urokinase conjugate showed 1,000 times higher activity than urokinase.
Several proteins with anticoagulatory activity have been isolated from human placenta. proteins were found to be members of the lipocortin/annexin family end to date, eight members of the family have been isolated from various tissues and cultured cells with many different functions proposed. The proteins are given the common name, "annexin". All annexins share the property of calcium-dependent binding to anionic phospholipids. Funakoshi et al., Biochem. (1987a) 26, 5572-5578; Tait et al., Biochem. (1988) 27, 6268-6276; R omisch and Helmburger, Biol. Chem. Hoppe-Seyler (1990) 371, 383-388.
Annexin V (also known as PAP-I) is a major component of the family and is isolated from placenta. It contains one free sulfhydryl group and does not have any attached carbohydrate chains. The primary structure of annexin V deduced from the cDNA sequence shows that annexin V comprises four internal repeating units (each unit has 60-80 amino acid residues). EPA 0 279 459; U.S. Pat. No. 4,937,324; Funakoshi etal., Biochem. (1987b) 26, 8087-8092. Among annexins, annexin V has the strongest binding affinity (K.sub.d &lt;10.sup.-10 M) for phospholipid vesicles containing 80% phosphatidylcholine (PC) and 20% PS under conditions that are comparable to plasma and extracellular fluid (1.2 mM ionized calcium, 0.15M ionic strength). Annexin shows high affinity for membranes containing PS and phosphatidic acid (PA), phospholipids carrying two negative charges. Tait & Gibson (1990) Cytekines Lipocortin Inflam. Diff., pp. 173-181. Binding is reversible and completely calcium-dependent.
Annexin V binds to human platelets. Unstimulated platelets express a small number of binding sites, but the number of binding sites is increased greatly by certain platelet agonists (for example, approximately 15-20 fold by a combination of thrombin and collagen). There are approximately 100,000 binding sites per platelet after stimulation with thrombin and collagen. The binding sites have an apparent dissociation constant (K.sub.d) of 7 nM. The binding is calcium-dependent, reversible and can be inhibited completely by PS-containing vesicles. Annexin V also can displace previously bound factor Xa from the platelet surface. Thiagarajan & Tait, J. Biol. Chem. (1990) 265, 17420-17423.
Annexin V inhibits all of the activation reactions in the coagulation cascade where phospholipid is involved. To catalyze the reactions, gamma-carboxyglutamic acid-containing coagulation factors bind to negatively charged phospholipids in the presence of Ca.sup.++. The dissociation constants of the .gamma.-carboxyglutamic acid-containing coagulation factors for phospholipid are in the 10.sup.-6 -10.sup.-7 M range, which is three to four orders of magnitude weaker than that of annexin V. (For the purposes of the instant invention, greater dissociation constants are those with greater numerical molarity values, thus a constant of 10.sup.-8 M is greater than a dissociation constant of 10.sup.-10 M. But of course, fort he present invention, a dissociation constant of 10.sup.-10 M represents greater binding propensity than a constant of 10.sup.-8 M). The inhibition mechanism of annexin V is to compete with the coagulation factors for binding to anionic phospholipids.
Levels of annexin V in human plasma and cells in contact with blood were measured by ELISA using an affinity purified rabbit antiserum. Annexin V is present intracellularly in platelets, endothelial cells and leukocytes but is absent in erythrocytes. Annexin V essentially is not present in normal human plasma and can be released by cell damage or death. Thus the protein that appears to be intracellular under most normal conditions can be released into the extracellular milieu with cell damage or death. Flaherty et al., J. Lab. Clin. Med. (1990) 115, 174-181.
In order to further improve fibrinolytic agents, a higher binding affinity for thrombi is desirable. The affinity of annexin V for negatively charged phospholipids is approximately 50 times stronger than that of tPA for fibrin and 10-100 times stronger than that of fibrin-specific antibodies.